In recent years, a number of in vitro methods have been developed for screening polypeptides for a desired binding specificity. For example, the phage display technique screens polypeptides displayed as a coat protein from a bacteriophage. See, e.g., Dower et al., WO 91/17271; McCafferty et al., WO 92/01047.; Ladner, U.S. Pat. No. 5,223,409 (incorporated by reference in their entirety for all purposes). Another in vitro screening method has been developed for isolating nucleic acid sequences that bind to proteins. See Gold et al., U.S. Pat. No. 5,270,163.
In vivo methods for screening libraries have also been reported. Such methods usually detect protein-protein or protein-nucleic acid interactions using reporter constructs to identify active members of a library (Allen et al., 1995). For example, a yeast three-hybrid system has been used to identify a cDNA encoding a protein that binds the 3' end of histone mRNAs, and bacterial reporter systems based on transcriptional antitermination or translational inhibition have been used to screen RNA-binding libraries in several different contexts (Fouts et al., 1996; Harada et al., 1996, Jain et al., 1996; Wilhelm et al., 1996). These methods are especially useful for screening cDNA expression libraries and, arguably, mimic physiological conditions more closely than the in vitro methods. Further, such methods allow screening for physiological or functional properties as distinct from merely binding activity. In vivo screens are usually performed in bacteria or fungi, such as yeast, because of high transformation efficiencies and because it is possible to transform many cells and obtain individual clones.
Because many libraries are screened to obtain therapeutic compounds and mammalian cells more closely simulate the environment of intended therapeutic use than procaryotic, yeast or in vitro screens, it would be desirable to screen libraries in mammalian cells. For example, protein folding or posttranslational modification of some peptides may be different in eucaryotic and procaryotic cells. Screening in mammalian cells is, however, generally more difficult than screening in procaryotes because: (1) transfection efficiencies are lower; (2) unlike bacteria or yeast where plasmid segregation results in clonal colonies, transfection of mammalian cells is believed to involve the uptake of a large population of plasmids; (3) in general, eucaryotic cells do not support episomal replication of plasmids, (4) establishment of stable eucaryotic cell lines with integrated vector is laborious and may not result in expression of many library members, (5) recovery of a selected vector from eucaryotic cells can be difficult.
Some progress has been reported to address these difficulties. Transfection efficiencies have been reported to be improved using liposomes, protoplasts, or retroviruses as delivery vehicles (Schaffner, 1980; Sandri-Goldin et al, 1981; Rassoulzadegan et al., 1982, Felgner et al., 1987; Kitamura et al., 1995). Selected plasmids have been reported to have been enriched from pools introduced into a cell by transfection by subdividing active pools and performing multiple rounds of enrichment (Seed, 1995). Further, plasmid recovery has been reported to be improved using episomally replicating plasmids containing SV40, polyoma, or Epstein Barr Virus (EBV) origins in specific cell types supporting episomal replication of such plasmids (Yates et al., 1985; Seed et al., 1987; Kinsella et al., 1996). Thus, for example, a cDNA expression cloning strategy has been reported by Seed et al., 1987 using plasmids containing an SV40 origin. This origin allows the plasmids to replicate episomally to high levels in COS cells, which express large T-antigen. SV40-vector libraries, were introduced into COS cells by protoplast fusion, the libraries were amplified episomally, and cells expressing the CD2 surface antigen were isolated by panning with antibodies, and amplified vectors were recovered from Hirt supernatants of the isolated cells.
Despite some progress as noted above, difficulties and limitations remain in screening libraries in eucaryotic cells, and improved methods are needed.